Research Plan: Diffuse glioma is currently incurable. Despite maximal treatment, the disease invariably relapses due, in part, to its ability to evolve. Thus, an important barrier to curative glioma treatment is the plasticity of malignant cells. Recent studies have shown that intra-tumoral heterogeneity fuels glioma evolution. Glioma progression relies on cancer stem cells (CSCs). These cells represent the tumor re-populating force and reconstitute the diverse hierarchy of cells composing the bulk of the tumor. In addition, CSCs may be more resistant to existing anti-cancer therapies. Nevertheless, glioma cellular phenotype and its fitness for selection result from both genetic and epigenetic alterations. Therefore, a major challenge in the study of glioma evolution is to integrate genetic and epigenetic heterogeneity. To overcome this challenge, we developed novel multi- modality single-cell sequencing technologies that enable direct integration across genetic, epigenetic, and transcriptional dimensions of cell-to-cell variation. By leveraging this innovative single-cell genomics toolkit, I will tackle the integration of genetic, epigenetic, and phenotypic heterogeneity across glioma primary clinical samples to systematically dissect drivers of cellular states that underlie glioma differentiation and evolution. First, relying on the ability to jointly genotype and transcriptionally profile thousands of glioma cells, I will define how cellular states (e.g., CSCs vs. more differentiated cells) impact the transcriptional output of subclonal mutations (Aim 1). Second, to test the hypothesis that epigenetic diversification significantly contributes to phenotypic diversity of gliomas, I will assess the role of dysregulation of epigenetic mechanisms in transcriptional programs of gliomas, and identify regulators (e.g., transcription and chromatin-modifying factors) likely to drive CSCs towards defined malignant cellular states by performing CRISPR knock-out experiments in glioma cells (Aim 2). Third, leveraging single-cell phylogenetic inferences and mathematical modeling of glioma plasticity, I will link differentiation hierarchies and cellular plasticity with lineage histories of glioma cells, defining plasticity in cell-state identity and its relation to genotype and epigenotype. These data will address the central question of the directionality of glioma differentiation (i.e., how likely are differentiated glioma cells to revert to CSCs), validated through experimental differentiation assays (Aim 3). There are no therapeutic strategies currently available to curb the evolutionary adaptation of glioma. Thus, these studies may pave the way for the future design of novel and improved therapeutic strategies aimed at targeting a specific glioma cellular phenotype. Career Development Plan: I have outlined a 5-year career development plan to meet my goal of becoming an independent investigator in cancer biology. I have assembled a Mentorship committee composed not only of leaders in the field but also of those able to directly impact my career advancement. Finally, Weill Cornell Medicine/New York Genome Center constitutes the ideal environment for attaining my scientific and career goals, given their outstanding research communities and track records of training independent scientists.